Production of a plant resistant to low temperatures

ABSTRACT

This invention relates to a DNA fragment comprising a base sequence (a) the base sequence referred to as nucleotide numbers 1-3794 in a sequence number 1 in a sequence list; (b) the base sequence (a) a part of which is deleted or substituted by another base sequence, or to which another base sequence is added. Further, the invention relates to a recombinant DNA and transformed plant including the above fragment. Moreover, the invention relates to fragment developing a promoter activity with a responsive property to low temperatures which comprises a base sequence (c) the base sequence referred to as nucleotide numbers 1-2797 in a sequence number 1 in a sequence list; (d) A part of the base sequence (c) developing a promoter activity with a responsive property to low temperatures; or (e) The base sequence (c) or (d) a part of which is deleted or substituted by another base sequence, or to which another base sequence is added. Further, the invention relates to a recombinant DNA and transformed plant including the above fragment.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] This invention relates to a DNA fragment, a recombinant DNA suitable to produce a breed such as a maize or rice having a low temperature resistance, and a transformed plant.

[0003] 2. Description of Related Art

[0004] From so far studies, it is found that there is a significant relationship between a low temperature resistance of a plant and a degree of unsaturation of fatty acid constructing a biomembrane thereof.

SUMMARY OF THE INVENTION

[0005] The inventors experimentally indicated that a transformed tobacco plant comes to have a higher resistance against low temperatures when a fatty acid unsaturating enzyme gene FAD7 derived from an arabidopsis is highly expressed in the plant.

[0006] When a production of a certain protein in a plant cell is required, a promoter operating constitutively and having a high promoter activity has been used. Such a promoter operates continuously, which is futile in the plant.

[0007] For instance, the constitutive promoter is also employed when a protein giving a plant a low temperature resistance is phenotypically expressed. Therefore, even at ordinary temperatures, the protein for giving the plant the low temperature resistance is forced to be unnecessarily expressed, causing unfavorable results.

[0008] For such reasons, a development of a site-specific and inducible promoter has been required. Particularly, it is required that an expression of a specific gene is strengthened only at low temperatures, in order to produce a breeding intermediate mother body in which a gene expressing an enzyme unsaturating a fatty acid and other protein genes contributing to a low temperature resistance is inducibly expressed in response to low temperatures, or in order to make possible production of an unstable functional protein using a plant cell.

[0009] It is, therefore, an object of the invention to produce a promoter capable of strengthening expression of a fatty-acid-unsaturating-enzyme gene and other protein genes contributing to a low temperature resistance in response to low temperatures.

[0010] Another object of the invention is to produce a breed having a low temperature resistance using the above promoter.

[0011] Still another object of the invention is to utilize the promoter in an inducibly generating system of a functional protein in a plant cell being specific to low temperatures.

[0012] According to a first aspect of the invention, there is the provision of a DNA fragment comprising a base sequence (a) or (b) described below:

[0013] (a) The base sequence referred to as nucleotide numbers 1-3794 in a sequence number 1 in a sequence list;

[0014] (b) The base sequence (a) a part of which is deleted or substituted by another base sequence, or to which another base sequence is added.

[0015] According to an aspect of the invention, there is the provision of a recombinant DNA and transformed plant having a low temperature resistance and induced a recombinant DNA including the DNA fragment mentioned above, respectively.

[0016] According to a second aspect of the invention, there is the provision of a DNA fragment developing a promoter activity with a responsive property to low temperatures, which comprises a base sequence (c), (d), or (e) described below:

[0017] (c) The base sequence referred to as nucleotide numbers 1-2797 in a sequence number 1 in a sequence list;

[0018] (d) A part of the base sequence (c) developing a promoter activity with a responsibility to low temperatures;

[0019] (e) The base sequence (c) or (d), a part of which is deleted or substituted by another base sequence, or to which another base sequence is added.

[0020] According to a second aspect of the invention, there is the provision of a recombinant DNA which includes the DNA fragment comprising the base sequence (c), (d), or (e) described above, and a transformed plant which transduces the recombinant DNA and phenotypically expresses a specific protein in response to low temperatures.

[0021] According to a third aspect of the invention, there is the provision of a DNA fragment developing a promoter activity, which comprises a base sequence (f), (g), or (h) described below:

[0022] (f) The base sequence referred to as nucleotide numbers 1-2271 in a sequence number 1 in a sequence list;

[0023] (g) A part of the base sequence (f) developing a promoter activity;

[0024] (h) The base sequence (f) or (g) a part of which is deleted or substituted by another base sequence, or to which another base sequence is added.

[0025] According to a forth aspect of the invention, there is the provision of a DNA fragment developing a responsive property to low temperatures, which comprises a base sequence (i), (j), or (k) described below:

[0026] (i) The base sequence referred to as nucleotide numbers 2272-2797 in a sequence number 1 in a sequence list;

[0027] (j) A part of the base sequence (i) developing a responsive property low temperatures;

[0028] (k) The base sequence (i) or (j), a part of which is deleted or substituted by another base sequence, or to which another base sequence is added.

[0029] Since a rice's low temperature inducible gene lip19 or maize's low temperature inducible gene mlip15 codes a DNA binding factor, they are considered to be a gene controlling transcription of other gene groups induced or repressed under low temperature stress. The inventors have clarified a functional unit of a low temperature inducible promoter in the maize's mlip15 gene and that the functional unit also operates in a transformed plant.

[0030] The inventors has isolated a genomic clone of the maize's mlip15 gene by an common method. Due to determination of nucleotide sequence, it becomes clear that the gene does not include a intron (see: Sequence number 1 in Sequence list).

[0031] In the gene mlip15, 2.8 kb of a nucleotide sequence (0.6 kb of a nontranslated region at 5′-end and 2.2 kb of a nucleotide sequence linking at upstream thereof, in a mlip15 cDNA) and 2.2 kb of a nucleotide sequence (the nucleotide sequence remained after subtracting 0.6 kb of the non-translated region at 5′-end from 2.8 kb of the nucleotide sequence) are respectively bound with a β-glucuronidase gene as a reporter gene to make recombinant DNAs.

[0032] Each recombinant DNA is introduced into a callus derived from a rice's scutellum by using a particle gun. The result is that the former maintains a reactivity for low temperatures but the latter lose it. Therefore, it has been clarified that the 2.8 kb fragment containing 0.6 kb of the nontranslated region at 5′-end has a promoter function responsive to low temperatures.

[0033] In the invention, as a vector used for producing a recombinant DNA, a plasmid can, for example, be employed. Further, as a plant used for introducing the recombinant DNA, a useful cultivating monocotyledon such as maize, rice, wheat, barley, oat, Italian millet, or Japanese millet is preferably used.

[0034] A protein produced by induction of the promoter according to the invention includes an ω-3-fatty acid unsaturating enzyme etc.

[0035] Moreover, the invention includes a base sequence one or several nucleotides of which are deleted or substituted by another base sequence, or to which another base sequence is added, if it maintains the promoter function according to the invention.

BRIEF DESCRIPTION OF THE DRAWING

[0036] The invention will be described with reference to the accompanying drawing, wherein:

[0037]FIG. 1 is an illustration diagrammatically showing mlip15 region.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] (Isolation of mlip15 genomic clone and determination of the nucleotide sequence)

[0039] A genome DNA prepared from a maize (breed: honey bantam) is cut (partially digested) by a restriction enzyme Sau3A and then separated by using a sucrose density gradient centrifugation to obtain 9.7-22 kb of DNA fraction. The DNA fraction is bound with a λ-EMBL3 digested by BamHI and then makes phage particles by using Giga-packGoldIIkit.

[0040] By examinating them by using Escherichia coli XL1-Blue MRA (P2) as a host, 1×10⁷ pfu/ml of a library is obtained.

[0041] The library is selected by using the whole length of the mlip15 cDNA as a probe to obtain three positive clones.

[0042] Among them, a clone named “λH1” is found to include 11.5 kb of a fragment containing the mlip15 cDNA, 5 kb of a fragment at the 5′-end, upstream thereof, and 5 kb of fragment at the 3′-end, downstream thereof.

[0043] In Sequence list 1 is shown 3,794 bp of a base sequence being an EcoRI-BamHI fragment including (consisting of) the whole length of the mlip15 cDNA.

[0044] A putative amino acid sequence at the region coding an mlip15 protein is symbolized under the base sequence by one capital letter. Nucleotide numbers corresponding to the assumed amino acid sequence are 2798-3205. A terminal codon of the base sequence is shown as a mark “*”. A nucleotide sequence which is assumed as “TATA box” is underlined and also has a description of “TATA box” thereunder. A transcription initiation point of mlip15 is T as nucleotide number 2272.

[0045] (Constructing of Recombinant DNA Used For Introducing to Rice'Callus)

[0046] A fragment having the nucleotide numbers 1-2797 and a fragment having the nucleotide numbers 1-2271 are respectively amplified by a PCR method (a polymerase chain reaction method) using the maize'mlip15 genomic clone as a template. The fragment of the nucleotide numbers 1-2797 is the above mentioned genomic sequence of 2.8 kb which consists of 0.6 kb of the nontranslated region at 5′-end and 2.2 kb of genomic sequence at upstream of the 0.6 kb of the non-translated region at 5′-end, in mlip15 cDNA . The fragment of the nucleotide numbers 1-2271 is the above mentioned genomic sequence of 2.2 kb and consists of the rest of 2.8 kb of the nucleotide sequence subtracted by 0.6 kb of the nontranslated region at 5′-end.

[0047] A nucleotide numbers 2798-3205 is a region coding the mlip15 protein.

[0048] As an enzyme in the above amplification, LATaqDNA polymerase having a proofreading activity is used. In this case, each primer is such designed that the base sequence of the nucleotide numbers 1-6 becomes a Hind III site (AAGCTT) and the base sequence of the nucleotide numbers 2792-2797 and the base sequence of the nucleotide numbers 2266-2271 respectively become a Bam HI site (GGATCC) to obtain each primer.

[0049] Each amplified fragment is integrated between the Bam HI and Hind III sites of a vector used for an Escherichia coli pUC18 and a confirmation of each base sequence is conducted. Using each plasmid with each base sequence confirmed, each Bam HI—Hind III fragment respectively consisting of 2797 bp and 2271 bp is reproduced and integrated between the Bam HI and Hind III sites of pBI 221. Each obtained recombinant plasmid is respectively named pBImp28 (because of containing 2.8 kb of promoter region in the mlip15) and pBImp22 (because of containing 2.2 kb of promoter region in the mlip15).

[0050] In FIG. 1, the mlip15 promoter region is diagrammatically illustrated. Beside, the mlip15 promoter portion contained in the above recombinant plasmid is shown therein. The symbol of “+1” indicates a transcription initiation point (the nucleotide number 2272), the symbol of “key arrow” the direction of transcription, and the symbol of “ATG” the position of a transcription initiation codon (the nucleotide number 2798). A β-glucuronidase gene (abbreviated as GUS) is used as a reporter gene (its scale is not correct in the figure).

[0051] (Reactivity of the Introduced Plasmid in Low Temperatures)

[0052] To a callus derived from a rice' scutellum (breed: Notohikari), the above each plasmid is introduced using a particle-gun (made by BIO-RAD Co., Ltd.) After the introduction, the callus is incubated for 24 hours in a dark place at 25° C. and then divided to two parts homogeneously. After respectively incubating for another 24 hours one at 25° C. and the other at 5° C., a GUS test is carried therefor. A GUS activity is judged as whether a low temperature reactivity is present or not, by calculating a ratio of the number of blue spots per scutellum on an X-Gluc as substrate at 5° C. to that of 25° C. (see: Table 1). Three times of tests are repeated on both plasmids and the results are shown in Table 1. TABLE 1 Induced plasmid $\frac{{Responsive}\quad {property}\quad {to}\quad {low}\quad {temperatures}}{{Ratio}\quad {of}\quad {GUS}\quad {actiity}\quad \left( {5{^\circ}\quad {{C.}/25}{^\circ}\quad {C.}} \right)}$

pBImp28 Experimental 1 2.7 Experimental 2 6.0 Experimental 3 4.7 pBImp22 Experimental 1 1.1 Experimental 2 0.62 Experimental 3 0.57

[0053] When the pBImp28 is introduced, a high GUS activity is obtained. But, when the pBImp22 is introduced, the GUS activity is inferior thereto. The following may be derived from these results.

[0054] (a) In DNA fragment of the nucleotide numbers 1-3794 of the maize' mlip15 gemonic clone, the promoter region relating to the low temperature responsive property exists at upstream of the putative amino acid sequence coding the mlip15 protein.

[0055] (b) The fragment of the nucleotide numbers 1-2797 contains the promoter region relating to the low temperature responsive property.

[0056] (c) The fragment of the nucleotide numbers 1-2271 is expected to effect as the promoter, but the fragment itself has a low responsive property to low temperatures. The fragment itself of the nucleotide numbers 2272-2797 has the low temperature responsive property and relates to the expression of the promoter.

1 2 1 3794 DNA Maize breed of honey bantam CDS (2798)..(3205) 1 gaattccgaa taacgcgccc cgcatgcaac cagatagcgg atctttcggc gctaaactca 60 gagggaagca attgccggaa gagtcggcgt gcaagaataa cataagtaga taagatttca 120 cgatctataa aaggatatct ccctagtcgg ctatataagg ctagggaggt acccaaacaa 180 aacgaatcac tctctttcac caccataacg cccactagta gactaatatg agatctcatc 240 caccgtcacc cgcgaatcat ctgtaaccca agcaaactca atacccaaca tcacacatga 300 cttagggtat tacgcattta ggcgacccga acctgtataa ttttcttgtg tttcaacgtg 360 cacctgcacg taccatcgag ttgcgattaa cgtcgccgtc ctcccaaaaa tactggtggt 420 cctcccaaaa actcgaccgg acgatagaaa tagtgtatgg ctaagtagag caaggtggcg 480 tttggtccgc ggctatgatg agaatccagt agactgaatc cacgtgctaa accaaacatc 540 actggttttg gctgcatctt ctcggattac gtgtgttgtg caaattctca taatccacgg 600 caaccagacg ggggcgcaac caattggtgt ttcttaggaa gcccccgcgt tacattggat 660 cataggatga ttcaagggtt atgatttttt agctactaat tggttgtcat catggtttat 720 aggtgaagat tgttattcaa tcaaagggcg acatatccct ccgcgtttag agacttgcgt 780 gtagtgtaaa catggatgta attgtgctac ctttaataga gtcccttagc tcttcaaaaa 840 caaatcttat tatatattaa ttactagtcc atccatttta ttctaattta gtttcgaaat 900 tactaaatat agaaaataaa atagagtttt agtagcaatt atgaaaactg aaatatagtt 960 ttaatttccg tatttagtga tttaaatact aaaatataat aaaatggaga gactaaaaac 1020 tagtccctat aaaccaaaca ttctttaaat aaagccccgt ggctaggaca atgacctatt 1080 tttttctcgc aaccggaaga ataaaaaatt caccgtaact ttctttcttt cttcttttta 1140 tgcgaaagaa gatagttgca agacgaatcc agagtttatc tggaagaaga aagttcctaa 1200 tcctcctcct tccctgtaga tattatcagc aaggcaagcg tgtcacggct tcttgcttga 1260 gtaatccgct cctatttttt tttttgggag ggcgccttcc taccggcttc gcttctaaac 1320 ggtgggcaaa tttggtacga taaagaaaaa agaggaggac gagtgggagg gcacttctgg 1380 aaaaaacttt ttaatgagct ggaccaagca gctgggcaag ctgtcactag gactggacaa 1440 aatactcgtg gctcgataac tcgctcgact cggctcgtta gtagctcagc tcgactcggc 1500 tcgttttaat tttgtagcga gccaagctag cattctagct cgattctcta atgagccagc 1560 tcgggttagc tcgtgagcta gctcgcgagc caaacgagct aagccacaac acaaatttgt 1620 ctagtcattg atgtcgtctc atctctcata gtcttgtttt ctcgtagtta tgatctgtga 1680 tatggacatg tgtggatgtg ccatgtgctt aaatatttat attattgcat ggctacatgt 1740 ttgtagtgtt aaatacttaa aatataattt ttcggttata aatatattta tgtacataga 1800 tatttatatt tagttgtgtg gctcacgagc ctaacgagct ggctcgagct tcctaacgag 1860 ccgagccgag ccagctattt agctcgttag tataacgagc cgagccgagc tggctcgtta 1920 tagtaacgag ccataacgag ccgagccata acgagccaag ctggttcgat atccacccct 1980 agctgtcacc gtcgcccagt ccgcttcgtt cggtcagcgg gccccacctc atctgcattc 2040 ttccattctc gtcctccgac ctcatctgca ttttcccagc caagtagtag gtaaactagt 2100 ggcggtcccg tggccgtggc atcaggaaaa gaatatgccg tcccagccca ccatcccccc 2160 accgtcccga aattccagaa ctaccctcgg ctccagctat aaatagccgc ccccgggaga 2220 cgttcgaaac cttccccatc tccggataaa agataaggag tgtctctcct ctctttcagc 2280 taagtccctg ctccctctct ttttcttaca ttcaggtcct cgcagctcct ctcttttttc 2340 ttgtttcttt ctttcgatct gcgagccgtc caggtccagt actctccttt ccgtgaagga 2400 actcttgcag ccggcccctc tggtttcctc gaattcttgt tccccggtcc ctcctcctgt 2460 ccccgcgtag atccgtccgt ccgaggagca caccgtcccc acccccatgt ttacccacca 2520 gttcctctga cggccgccgt gctccgatga agctgagcgt gctccgtatc cgccgctccc 2580 actccttctc cgtcgccttc ctctactggt tctacgtctt ctcatgaacg catcgcccct 2640 ctccacctgc tgatccttcg ccatctctcc atctctcttt ctctctgaga tagtctttcg 2700 aatccatctc tagggctctt gtttctcccc atcctccccc caccccaccc cccaccaaac 2760 acaagtcccc ttgttcaatc cgacaagaca agcatcc atg tcg tcg tca cgc cgg 2815 Met Ser Ser Ser Arg Arg 1 5 agc tcg agc ccc gac agc aac gac acg acg gac gag cgc aag cgg aag 2863 Ser Ser Ser Pro Asp Ser Asn Asp Thr Thr Asp Glu Arg Lys Arg Lys 10 15 20 cgg atg ctg tcc aac agg gag tcg gcg cgg cgg tcg cgc gcg cgg aag 2911 Arg Met Leu Ser Asn Arg Glu Ser Ala Arg Arg Ser Arg Ala Arg Lys 25 30 35 cag cag cgg ctg gag gag ctg gtg gcg gag gtg gcc cgc ctg cag gcg 2959 Gln Gln Arg Leu Glu Glu Leu Val Ala Glu Val Ala Arg Leu Gln Ala 40 45 50 gag aac gcg gcg acg cag gcc cgc acc gcg gcg ctg gag cgc gac ctg 3007 Glu Asn Ala Ala Thr Gln Ala Arg Thr Ala Ala Leu Glu Arg Asp Leu 55 60 65 70 ggc agg gtg gac ggc gac aac gcg gtc gtg cgc gcc cgc cac gcc gag 3055 Gly Arg Val Asp Gly Asp Asn Ala Val Val Arg Ala Arg His Ala Glu 75 80 85 ctg gcc ggc cgc ctg cag tcg ctg ggc ggc gtc ctc gag gtg ctc cag 3103 Leu Ala Gly Arg Leu Gln Ser Leu Gly Gly Val Leu Glu Val Leu Gln 90 95 100 atg gcc ggc gcc gcc gtc gac atc ccg gag atg gtc acc gac gac ccc 3151 Met Ala Gly Ala Ala Val Asp Ile Pro Glu Met Val Thr Asp Asp Pro 105 110 115 atg ctc cgc ccc tgg cag ccg tcc ttc ccc ccg atg cag ccc atc ggg 3199 Met Leu Arg Pro Trp Gln Pro Ser Phe Pro Pro Met Gln Pro Ile Gly 120 125 130 ttc tga gaatctgagc ctcagccggc gggagagagc caatttctgt cgtcgtgccg 3255 Phe 135 ctgtctatct cgtattggta tatctattca taaatcatcc ttgtcatggt ttggtcttct 3315 tgttcagtgt tataaatttg cttcttgtta gtgttataaa tttggccatc ggaaaggatg 3375 tgtttgtagt tgtaatatct tgtttggagt tgtaatatct tatcttgctt atgaaatcga 3435 atatgcctat atatatatgt tatgctgtac gagtatgtgg ctccaaattt gtgagccttc 3495 tgtctgttat ggtgaggcga tgaatccaat ttgtgagcac acatgaatca atttcgagat 3555 tcgacatgtc aagttgatcg ttgcaggaag gacggttttt gtatggacgg acataccaag 3615 ttactgcatt ttacttaaaa tatctcactt attttttaga tcggcatttc tccactcgtt 3675 agatttcttg ttcttgagtc agaagataac tacagcatgt catatctcaa ttggaatacc 3735 attagggtcc ctcatcttaa cctatttcat ctcttttaat acgtagattt tttggatcc 3794 2 135 PRT Maize breed of honey bantam 2 Met Ser Ser Ser Arg Arg Ser Ser Ser Pro Asp Ser Asn Asp Thr Thr 1 5 10 15 Asp Glu Arg Lys Arg Lys Arg Met Leu Ser Asn Arg Glu Ser Ala Arg 20 25 30 Arg Ser Arg Ala Arg Lys Gln Gln Arg Leu Glu Glu Leu Val Ala Glu 35 40 45 Val Ala Arg Leu Gln Ala Glu Asn Ala Ala Thr Gln Ala Arg Thr Ala 50 55 60 Ala Leu Glu Arg Asp Leu Gly Arg Val Asp Gly Asp Asn Ala Val Val 65 70 75 80 Arg Ala Arg His Ala Glu Leu Ala Gly Arg Leu Gln Ser Leu Gly Gly 85 90 95 Val Leu Glu Val Leu Gln Met Ala Gly Ala Ala Val Asp Ile Pro Glu 100 105 110 Met Val Thr Asp Asp Pro Met Leu Arg Pro Trp Gln Pro Ser Phe Pro 115 120 125 Pro Met Gln Pro Ile Gly Phe 130 135 

What is claimed is:
 1. A DNA fragment comprising a base sequence (a) or (b) described below: (a) The base sequence referred to as nucleotide numbers 1-3794 in a sequence number 1 in a sequence list; (b) The base sequence (a) a part of which is deleted or substituted by a base sequence, or to which a base sequence is added.
 2. A DNA fragment according to claim 1, wherein the DNA fragment includes a maize' low temperature inducible gene mlip15
 3. A DNA fragment developing a promoter activity with a responsive property to low temperatures, which comprises a base sequence (c), (d), or (e) described below: (c) The base sequence referred to as nucleotide numbers 1-2797 in a sequence number 1 in a sequence list; (d) A part of the base sequence (c) developing a promoter activity with a responsibility to low temperatures; (e) The base sequence (c) or (d) a part of which is deleted or substituted by another base sequence, or to which another base sequence is added.
 4. A DNA fragment developing a promoter activity which comprises a base sequence (f), (g), or (h) described below: (f) The base sequence referred to as nucleotide numbers 1-2271 in a sequence number 1 in a sequence list; (g) A part of the base sequence (f) developing a promoter activity; (h) The base sequence (f) or (g) a part of which is deleted or substituted by another base sequence, or to which another base sequence is added.
 5. A DNA fragment developing a responsive property to low temperatures, which comprises a base sequence (i), (j), or (k) described below: (i) The base sequence referred to as nucleotide numbers 2272-2797 in a sequence number 1 in a sequence list; (j) A part of the base sequence (i) developing a responsibility to low temperatures; (k) The base sequence (i) or (j) a part of which is deleted or substituted by another base sequence, or to which another base sequence is added.
 6. A recombinant DNA comprising a DNA fragment as claimed in claim
 1. 7. A recombinant DNA comprising a DNA fragment as claimed in claim
 3. 8. A transformed plant having a low temperature resistance in which the transformed plant transduces a recombinant DNA including a DNA fragment as claimed in claim
 1. 9. A transformed plant phenotypically expressing a specific protein in response to low temperatures, in which the transformed plant transduces a recombinant DNA including a DNA fragment in claim
 3. 